The field of this invention relates to the introduction of DNA segments, which may be genes, into bacterial plasmids, and the use of the resulting recombinant plasmids for bacterial cloning of the plasmids and/or expression of the proteins directed by the recombinant DNAs. In particular, this invention is concerned with the preparation of plasmids containing many tandem copies of a selected DNA segment. Since genes produce their products (proteins) in approximate proportion to their number (see, for example, S. Normark, et al, J. Bacteriol. 132, 912922, 1977), plasmids containing many copies of a protein gene can be used to cause bacteria to make that protein in large quantities. This invention is thus an extension of recombinant DNA technology. In addition, the multiple copies of the DNA segments have commercial value in themselves, for example as reagents for recombinant DNA processes.
J. R. Sadler and colleagues (Sadler, et al, Gene, 3, 211-232, 1978) were able to prepare from the plasmid pMB9 recombinant plasmids containing from one to four tandem repeats of a synthetic lac operator. (An operator in this context is a DNA segment which controls the expression of a gene.) Later attempts by Sadler and his associates to prepare plasmids containing larger numbers of copies of the operator employed the plasmids containing four operators as starting material (Sadler, et al, Gene, 8, 279-300, 1980). By a laborious procedure they were able to prepare a plasmid containing 12 lac operators. One serious problem was the in vivo instability of the plasmids containing multiple operators. It was reported that a primary cause of the instability was believed to be inverted orientations of some of the operator segments in the recombinant plasmids. Such inversions in plasmids containing three or more operators were said to lead to rapid reduction, in vivo of the number of operators, usually to 1 or 2 operators.
With reference to the work of Sadler, et al, which is believed to represent the state of the art in this field, there is a clear need for simpler and more direct procedures for preparing plasmids containing multiple copies of DNA segments, and particularly for plasmids with the repeated sequences arranged uniformly in head-to-tail conformation without segment inversions causing in vivo instability.